Conformational dynamics play essential roles in the functions of non-coding RNAs (ncRNAs), including gene regulation by riboswitches, enzymatic catalysis by small self-cleaving ribozymes, and protein synthesis by ribosomes. The conformational changes can take place in multiple steps, each triggered by distinct cellular inputs and lead to distinct structures that serve unique functions during the multi-step biochemical pathways. Therefore, an integrated structural, dynamic, thermodynamic, and kinetic view of ncRNAs is crucial for developing deep understanding of their biological mechanisms. Riboswitches and ribozymes are two classic ncRNAs that serve as protein-independent regulators in critical cellular processes such as transcription and translation. Here, we propose to develop a deep and comprehensive understanding regarding the dynamic mechanisms of these two classes of ncRNAs, using the fluoride riboswitch and the hammerhead ribozyme as model systems to exemplify RNA's regulatory and catalytic activities. Towards our long-term goal of elucidating how riboswitches control gene expressions and how ribozymes perform catalysis, the overall objective of this proposed research is to develop and apply solution NMR methods to visualize the free energy landscapes that govern their mechanisms and to perform biochemical assays and mutagenesis to reengineer individual functional steps to test predictions. To accomplish this overall objective, the proposed research details three specific objectives that feature a gradual increase in the complexity of structure and dynamics: (1) delineate the mechanism of ligand binding of the fluoride riboswitch aptamer, (2) characterize the signal transduction between the aptamer and the expression platform of the fluoride riboswitch, and (3) elucidate the role of allosteric conformational dynamics in enzymatic catalysis by the hammerhead ribozyme. Results will be used to examine the central hypothesis of this proposal that RNA structures have evolved to encode complex conformational landscapes to direct structural changes along specific functional pathways. These proposed studies will facilitate developing a better mechanistic understanding of riboswitch and ribozyme functions and formulating foundations for studying even more complex riboswitches and ribozymes. Understanding how riboswitches and ribozymes work will further assist the development of riboswitch- targeted antimicrobial therapeutics, ribozyme-based gene knockdown tools, and de novo design and precise engineering of novel RNA functions. The proposed high-resolution NMR methods will also provide the field with tools and techniques for advancing the molecular understanding of other ncRNA functions.